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Acta Physiologiae Plantarum ISSN 0137-5881Volume 34Number 6 Acta Physiol Plant (2012) 34:2419-2424DOI 10.1007/s11738-012-1009-8

Influence of the irradiance on phenolscontent and rooting of Ilex paraguariensiscuttings collected from adult plants

José Tarragó, Roxana Filip, LuisMroginski & Pedro Sansberro

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SHORT COMMUNICATION

Influence of the irradiance on phenols content and rootingof Ilex paraguariensis cuttings collected from adult plants

Jose Tarrago • Roxana Filip • Luis Mroginski •

Pedro Sansberro

Received: 3 May 2011 / Revised: 23 April 2012 / Accepted: 25 April 2012 / Published online: 22 May 2012

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2012

Abstract The influence of irradiance on phenolics con-

tents and rooting of Ilex paraguariensis cuttings was

studied. Results of the first experiment with stock plants

under controlled-irradiance conditions show that when the

irradiance level increased from 1.5 to 100 % PPFD, the

oxidation of cuttings raised from 19 ± 11 to 88 ± 4 %

(r2 = 0.64). At the same time, a strong correlation was

observed between total phenolics content and irradiance

(r2 = 0.7). In consequence, adventitious rooting dimin-

ished from 67 ± 5 to 3 ± 3 % under full radiation

(r2 = 0.7). In the second experiment with stock plants

subjected to field conditions, the results showed that the

rooting process is strongly affected by the genotype

(P \ 0.0001), while the statistical analysis did not show a

correlation between rooting and age of the donor plant.

Season had a variable effect and depends on genotype.

Although we did not find correlations between the rooting

ability and the canopy structure of the stock plants, the

position of the branches in the mother plant affected

rooting and depended on season in addition to genotype.

Concomitantly, the levels of soluble phenolics compounds

were higher from leaves subjected to high-irradiance con-

ditions than samples collected from inner canopy; which

was coincident with the pattern of cuttings oxidation. In

conclusion, our results provide evidences which support

the hypothesis that the physiological status of the stock

plant at the time that cuttings are excised is of utmost

importance for the subsequent rooting of I. paraguariensis

cuttings. The influence on soluble phenolics content of

different irradiances given to the stock plants negatively

affect the rooting process since the product of its oxidation

cause the browning and death of the cuttings.

Keywords Ilex paraguariensis � Stock plant �Adventitious rooting � Soluble phenolics content

Introduction

Ilex paraguariensis St. Hil. is the most cultivated specie of

the genus Ilex in America due to its economical relevance.

Its leaves and shoots are used to prepare a traditional

infusion named mate which has several health benefits. The

establishment of a useful method for vegetative propaga-

tion of mature trees is difficult due to the reduced rooting

capacity of softwood cuttings. However, it is not known

whether the characteristics of cuttings are associated with

genetic differences in rooting ability or simply an expres-

sion of growth and the condition of plant since the material

for cuttings is normally sourced from established stock

plants exposed to seasonal changes and subjected to a

variety of environmental stresses which influence the

growth and ability to provide cutting material that forms

adventitious roots (Kibbler et al. 2004). Rooting success

has often been correlated with the temperature of the stock

plant environment prior to take cuttings. However,

increased rooting may not be only the result of high tem-

perature per se since it should be influence by the

Communicated by K. Trebacz.

J. Tarrago � L. Mroginski � P. Sansberro (&)

Instituto de Botanica del Nordeste (IBONE-CONICET),

Facultad de Ciencias Agrarias (UNNE), Sgto. Cabral 2131,

CC: 209, W3402BKG Corrientes, Argentina

e-mail: sansber@agr.unne.edu.ar

R. Filip

Catedra de Farmacognosia, Facultad de Farmacia y Bioquımica,

IQUIMEFA (UBA-CONICET), CP: 1113,

Junın 956, Buenos Aires, Argentina

123

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DOI 10.1007/s11738-012-1009-8

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physiological state of the plant (Husen and Pal 2007). In

fact, it is well known that plants adjust the level of free

endogenous hormones and a variety of metabolites such as

soluble sugars (Rosa et al. 2009), phenols (Edreva et al.

2008), and flavonoids (Treutter 2006) to reduce the nega-

tive impact of the adverse situation. Environmental influ-

ence may act through the variation in the level of auxin and

certain phenols which affect the sensitivity to the rooting

stimulus exert by the auxin (Faivre-Rampant et al. 2002).

Numerous reports confirm the activity of ortho-dihydroxy

phenols as a rooting co-factor or synergist. For example,

p-coumaric, caffeic and chlorogenic acids enhanced root-

ing when supplied alone and increased the effect of

auxin applications (Jarvis 1986). Furthermore, our previous

result demonstrates that flavonoids promote rooting in

I. paraguariensis cuttings (Tarrago et al. 2004). We found

that quercetin promoted the formation of adventitious roots

and improved the distribution of roots around the cutting

without impacting the number of roots per rooted cutting.

In contrast, the additions of naringenin or rutin to the

culture medium inhibited the in vitro rooting of Ilex

dumosa micro-shoots (Luna et al. 2003). Concomitantly,

many plants naturally synthesize mono or polyphenolic

compounds in response to either biotic or abiotic stresses

which negatively affect the morphogenetic process

(Vermerris and Nicholson 2008).

The aim of this study was to determine whether

I. paraguariensis rooting correlates with the phenological

status of the mother plant, the sunlight conditions, the total

soluble phenols content, and the variation of chlorogenic

acid, dicaffeoylquinic acids, caffeic acid, and rutin in the

leaves at the time of cutting collections.

Materials and methods

Experiments

The first experiment aimed to determine whether irradiance

on the stock plants correlates with rooting. Cuttings were

obtained from stock plants of SI-49 clone grown in 4 L pots

filled with lateritic red soil and subjected to different sun-

light irradiances by covering the roof, eastern and western

sides of iron-framed boxes with different layers of shading

nets. Each box contained 12 pots and the light treatments

(boxes) were repeated three times. The environmental

conditions registered throughout the 120-day-experiment

were as follows: average temperature 26.8 �C (ranging

from 21.2 to 32.7 �C), average RH 65 % (ranging from 42

to 89 %). Only running tap water was added to the pots to

keep the soil moisture at field capacity. This experiment

was carried out in summer and cuttings were collected at

the end of the second flash of growth.

Based on experiment 1, the second experiment was

achieved to evaluate whether season and canopy structure

of the field stock plants have an effect on the rooting

process. Cuttings were collected from 15 mature plants

localized in Gdor. Virasoro, Argentina (288020S, 558540W).

The site is characterized by a mean annual rainfall of

1,800 mm, distributed mainly during spring and autumn;

mean year temperature of 22 �C while frosts are scarce.

The soil is described as Ultisol. Shoots (35 to 40-cm long)

were harvested from the central and peripheral structure of

each plant (inner and outer canopy) at the end of each

seasonal flash of growth from 14 to 78-year-old plant

grown at different densities.

Treatment of cuttings and growing conditions

Procedures for the collection and handling of cutting

material are described in Tarrago et al. (2004). Softwood

cuttings (10 to 12-cm long, 3 to 5-mm diameter) consisted

of six to nine nodes in which the uppermost mature leaf

was cut in half and retained while the lower six to eight

leaves were removed. For rooting, cuttings were dipped in

an aqueous solution of quercetin 500 lM (60 min) fol-

lowed by a treatment of 4 min in 4,000 ppm of IBA eth-

anolic solution (50 %) and set into trays containing perlite

plus 0.5 g of controlled release micro-fertilize (Osmoco-

te�, 18-5-9). They were grown for 6–8 weeks in a growth

chamber providing a day/night air temperature of 25–27/

20–22 �C and substrate temperature of 22–25 �C. Relative

humidity was maintained at 90 % during the first 7 days by

a fog device and then decreased gradually until 70 %.

Photoperiod of 12 h was kept throughout the rooting period

using 20 % sunlight radiation (150–180 lmol m-2 s-1,

PAR) plus 100 lmol m-2 s-1 using eight cool-white

fluorescent lamps (40 W) set at 1.8 m over the cuttings and

outside the growth chamber.

Analysis of phenolic compounds

Total polyphenol determination

Hundred and twenty-five milligram of frozen powder from

mature leaves was suspended in 500 ll methanol and

incubated at room temperature (10 min). After centrifu-

gation at 2,500g (10 min), 400 ll supernatant was col-

lected. The pellet was re-extracted under the same

conditions and 400 ll supernatant was removed and pooled

with the first supernatant. Afterward, the extract was ten

times concentrated by evaporation at 45 �C. To estimate

the amount of phenolics, the methanolic extracts were

diluted 1:40 with water and the Folin-Ciocalteu reagent

diluted 8 times with distilled water (200 ll) and Na2CO3

20 % (650 ll) were added to the samples. Tubes were

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incubated in darkness at room temperature for 120 min.

Absorbance was measured at 765 nm. Results were

expressed in gallic acid equivalent per milligram of leaf

samples.

High performance liquid chromatography

The quantification of caffeoyl derivatives was carried out

using validated HPLC external standard methods. A

reverse phase IB-SIL RP 18 (5 lm, 250 9 4.6 mm I.D)

Phenomenex column and a gradient consisting of solvent

A: water: acetic acid (98:2); solvent B: methanol: acetic

acid (98:2) was used. A gradient range from 15 % B to

40 % B in 30 min; 40 % B to 7 5% B in 10 min, and 75 %

B to 85 % B in 5 min was employed. Flow rate was set at

1.2 ml/min. Identification and quantification were carried

out by simultaneous analysis of retention times and

detection with an UV detector and a photodiode-array

detector at 325 nm for caffeoyl derivatives and 254 nm for

routine.

Statistical analysis

Each treatment consisted of 24 cuttings and the experi-

ments were repeated three times. Data were analyzed with

ANOVA and regression modules. For analyses of corre-

lations and lineal regression between PPFD values, repli-

cates were individually considered.

Results and discussion

Table 1 shows the results obtained from potted SI-49 plants

subjected to different sunlight conditions. Oxidation of

cuttings raised from 19 ± 11 to 88 ± 4 % when the irra-

diance level increased from 1.5 to 100 % PPFD

(r2 = 0.64). Likewise, a strong correlation was observed

between total phenols content and irradiance (r2 = 0.7).

Adventitious rooting diminished from 67 ± 5 to 3 ± 3 %

under full radiation (r2 = 0.7). Furthermore, the number of

roots per rooted cutting was negatively affected by the

incident radiation.

The rooting ability of cuttings taken from stock plants

subjected to field conditions is shown in Table 2. The

genotype strongly affected this morphogenetic process

(P \ 0.0001). Statistical analysis did not show a positive

relationship between rooting and age of the donor plant.

Season had a variable effect and depends on genotype. In

most cases, rooting was greater in spring and summer than

autumn (P \ 0.0001) with the exception of V-8. Although

we did not find correlations between the rooting ability and

the canopy structure of the stock plants when the density

varied from 1,000 to 5,600 plants per ha, the position of the

branches in the mother plant affected rooting and depended

on season in addition to genotype. In fact, rooting of

varieties A-5 and V-11 (78 and 17-year-old, respectively)

was higher in spring and the best result was obtained when

the explants were collected from inner canopy. Otherwise,

summer had a positive effect on the rooting process from

V-1, V-12, and V-16 with a further interaction between

canopy position and genotype. Although, V-1 and V-12

was the same age and belonged to the same orchard with an

equal density, the obtained results were disparities respect

to the position of the explants.

In general, the levels of soluble phenolics compounds

were higher from leaves subjected to high irradiance con-

ditions than samples collected from inner canopy and the

pattern of oxidation show a similar performance, except for

A-4, A-8, A-10, and V-5, in which, the results should not

be explained only by the total phenolics content. In addi-

tion, the endogenous content of some diphenols (chloro-

genic acid, dicaffeoylquinic acids) and flavonoids (caffeic

acid, rutin) extracted from leaves of different varieties

indicated that its whole concentration (Table 3) is not

linked with the rooting ability exhibit by the genotype and

its variation should be associated with the radiation level

Table 1 Influence of PAR

irradiance (PPFD) on the leaf

soluble phenolics content,

oxidation, and rooting of

cuttings obtained from stock

plants grown in pots

Each value is shown as the

mean ± SEM

PPFD (%) Linear

regression(r2–F)1.5 3 15 100

Leaf soluble

phenolics

(ppm eq. gallic

acid g-1 FW)

28,222 ± 4,821 19,524 ± 4,085 25,557 ± 3,586 47,797 ± 3,871 0.7–22.8

Oxidation and death

of cuttings (%)

24 ± 3.4 19 ± 11.1 67 ± 4.1 88 ± 4.3 0.6–17.6

Rooting (%) 67 ± 4.8 63 ± 8.9 25 ± 0.4 3 ± 3 0.7–22.0

Roots per rooted

cutting

10.1 ± 2.0 6.7 ± 1.3 5.3 ± 2.0 0.3 ± 0.3 0.6–14.5

Acta Physiol Plant (2012) 34:2419–2424 2421

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determined by the position of the branches in the donor

plant.

Our results indicate that the variation in rooting ability

was the result of genotypic difference and physiological

state of the stock plants. Adventitious root formation is a

culmination of the complex but specific response of some

genomic domain(s) of competent cells elicited by the

diverse effects of the external/internal environment (Ansari

and Singh 2008). However, the genetic control and asso-

ciated molecular mechanisms underlying adventitious

rooting are still largely unknown (Han et al. 2009). Haissig

and Riemenschneider (1988) arbitrarily categorized four

genetic effects responsible for the process as direct, cor-

related, uncorrelated, and regulatory. The first effect refers

to the genomic domain(s) carrying information for the

process. The remaining three effects are peripheral and

indirectly regulate the process via interactions with the

first, e.g., expression of other genomic domain(s) respon-

sible for synthesis and supply of metabolites and/or specific

regulatory molecules. Even if genotypic differences in

Table 2 Effect of branch position, season, genotype and their interactions on leaf soluble phenolics content, oxidation, and rooting response of

Ilex paraguariensis softwood cuttings taken from the field

Cultivar and

age (years)

Orchard stands

(plants/ha)

Canopy

structure

Branch

position

Soluble phenolics

ppm eq. gallic

ac g-1 FW

Oxidation of

cuttings (%)

Rooting (%)

Spring Summer Autumn

A-4 (78) 1,250 Open Interior 31,054 ± 1,586 25 ± 1.2* 24 ± 10.3 53 ± 1.4 17 ± 2.9

Periphery 65,857 ± 401*** 10 ± 5.2 12 ± 6.9 45 ± 2.6 22 ± 1.9

A-5 (78) 1,250 Open Interior 23,466 ± 13,816 31 ± 6.6 75 ± 6.9* 15 ± 2.1** 26 ± 4.9

Periphery 28,272 ± 3,950 30 ± 3.3 24 ± 2.6 55 ± 3.4 5 ± 5.0

A-6 (78) 1,000 Open Interior 30,779 ± 255 2 ± 2.0 11 ± 0.7 25 ± 1.8* 11 ± 5.5

Periphery 57,000 ± 9,026* 67 ± 2.9*** 10 ± 5.2 15 ± 0.4 10 ± 5.2

A-7 (78) 1,000 Open Interior 23,546 ± 2,881 28 ± 4.1 3 ± 3.0 0 0

Periphery 55,391 ± 10,384* 49 ± 8.9* 25 ± 5.5 16 ± 1.2** 10 ± 5.2

A-8 (78) 1,000 Open Interior 16,576 ± 9,120 70 ± 8.9* 16 ± 5.3 5 ± 2.5 37 ± 6.5

Periphery 34,062 ± 1,067 30 ± 2.0 36 ± 3.2* 56 ± 4.0** 37 ± 6.5

A-10 (78) 1,000 Open Interior 27,917 ± 5,169 48 ± 2.8*** 12 ± 2.6 12 ± 2.1 0

Periphery 34,228 ± 2,117 14 ± 1.4 1 ± 1.0 13 ± 1.6 0

V-1 (30) 1,250 Open Interior 24,493 ± 5,117 41 ± 6.9 35 ± 2.9* 21 ± 3.3 16 ± 0.8

Periphery 36,745 ± 2,566 40 ± 5.8 29 ± 2.1 52 ± 13.6 11 ± 5.5

V-2 (30) 1,250 Open Interior 22,516 ± 3,754 92 ± 8.3 28 ± 2.8 8 ± 8.0 11 ± 5.5

Periphery 34,399 ± 4,340 100 25 0 8 ± 8.0

V-12 (30) 1,250 Open Interior 24,263 ± 2,474 0 36 ± 7.3* 65 ± 0.8** 7 ± 7.0

Periphery 27,285 ± 2,783 22 ± 6.9 12 ± 3.2 3 ± 3.0 0

V-4 (25) 2,222 Open Interior 30,182 ± 642 10 ± 2.1 56 ± 1.4 72 ± 3.9 53 ± 7.0

Periphery 36,015 ± 1,455* 14 ± 7.5 49 ± 7.2 53 ± 1.5 30 ± 1.6

V-5 (27) 3,300 Middle Interior 27,322 ± 1,744 64 ± 5.7 25 ± 1.3* 8 ± 3.9 6 ± 2.9

Periphery 55,634 ± 3,758** 0 11 ± 3.3 32 ± 5.1 0

V-6 (18) 3,300 Middle Interior 30,309 ± 7,984 47 ± 4.6 20 ± 2** 7 ± 1.9 0

Periphery 51,533 ± 3,604 50 ± 4.1 0 0 0

V-11 (17) 3,300 Middle Interior 24,025 ± 3,346 7 ± 3.7 89 ± 5** 7 ± 3.5* 16 ± 0.8

Periphery 35,046 ± 1,892* 18 ± 5.1 44 ± 4.3 27 ± 1.2 10 ± 5.2

V-16 (17) 3,300 Middle Interior 21,836 ± 1,634 0 30 ± 3** 88 ± 6.5* 0

Periphery 35,683 ± 1,992** 25 ± 5.4 0 36 ± 7.2 0

V-17 (17) 3,300 Middle Interior 30,972 ± 2,513 30 ± 3.7 32 ± 6.1* 26 ± 1.2 20 ± 12.4

Periphery 32,508 ± 5,415 25 ± 9.1 3 ± 3.0 25 ± 5.7 26 ± 4.9

SI-49 (14) 5,600 Close Interior 12,834 ± 764 33 ± 9.5 NT 70 ± 1.6 NT

Periphery 25,418 ± 683*** 29 ± 6.4 NT 54 ± 4.2 NT

For leaf soluble phenolics determination, samples were collected from internal and peripheral branches at the end of the second flash of growth

(summer). Each value is shown as the mean ± SEM

NT not tested

*, **, and *** reflects significant difference between branch position at P \ 0.05, P \ 0.01, and P \ 0.001 level, respectively

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utilization of hormones and metabolism of proteins and

soluble carbohydrates available in the cutting likely con-

tributed to variable response (Husen 2008), genotype

x environment interactions are thought to have a major role

in governing rooting response (Zalesny et al. 2005). In this

study, the difficulty of separating environmental effects

from genetic differences was overcame by comparing

cuttings of a unique genotype grown in pots and subjected

to different irradiance conditions. Percent rooting was

negatively influenced by irradiance which probably deter-

mines a higher temperature and lower relative humid

conditions at the level of the canopy. Furthermore, light or

the exclusion of light can be a major factor that influences

the physiological and anatomical status of the stock plant

and the subsequent adventitious root formation of cuttings

collected from these plants (Pijut et al. 2011). In addition,

other factors like wounding, pathogens, symbiotic bacteria,

and development regulate the activity of enzymes that

control secondary metabolites biosynthesis such as phenols

and its derivate compounds which play a major role in the

adaptation of plants to the changing environment and in

overcoming stress constraints (Edreva et al. 2008).

Chemically, phenols are extremely heterogeneous sub-

stances and may range from simple monomers to very large

polymers which may combine with proteins, either

reversibly by hydrogen bonding or irreversibly by oxida-

tion, holding backs the enzyme activity (Croteau et al.

2000). Our result clearly showed a high correlation

between irradiance and leaf soluble phenolic content which

promote the declination and death of cuttings.

Considering that I. paraguariensis plants every year

have three periods of rapid shoot elongation, which alter-

nate with periods of little or no growth (flashing) wherein is

subjected to different environmental stresses such as high

temperature and water deficit (Sansberro et al. 2004), we

analyzed whether season and canopy structure of the field

stock plants affect the survival and rooting of cuttings. The

results of our experiments confirmed that the level of

soluble phenolic content was higher from leaves exposed to

full sunlight radiation than those collected from the central

zone of the plant subjected to a more suitable condition;

however, the leaf phenolics content not always show a

correlation with the browning of the cuttings which may be

explained at the base of the chemical heterogeneity of

phenolic structure that could be a stimulator or inhibitor

factor of adventitious rooting (Faivre-Rampant et al. 2002).

Phenolic biosynthesis is positively affected by the envi-

ronment (Ghasemzadeh and Ghasemzadeh 2011). It has

been demonstrated that mainly light and thermal stress

induces the production of flavonoids and phenylpropanoids

compounds in chloroplast and cytoplasm through phenyl-

alanine pathway, which is considered by most authors to be

one of the main lines of cell acclimation against stress in

plants (Rivero et al. 2001). Coumaric acid and caffeic acid

are derived from cinnamic acid and lead to secondary

metabolites such as flavonoids and phenolic acids. In the

later step, chlorogenic acid is synthesized from caffeic acid

and promotes the formation of dicaffeoylquinic acids

(Moglia et al. 2008). In some way, the variation of phen-

olics content observed in our experiments should be

explained based on its biosynthetic pathway as a clear

response of the genotype to the environment. For example,

while caffeic acid content decreased from 17 to 40 % in

external leaves subjected to stress, the level of chlorogenic

acid and its dicaffeoylquinic derived compounds varied

according with the genotype. Expectedly, chlorogenic acid

content increased in SI-49 clone by 40 % while the level of

dicaffeoylquinic acids decreased from 12 to 36 %. This

fact should be related with the mechanism of tolerance to

drought performed by this genotype (data not shown), since

Rivero et al. (2001) have reported the specific involvement

of chlorogenic acid in stress responses. The results of

current study suggest the ability of different shade level

forced by the branch position in altering or modifying both

the concentration and profiling of phenolics and flavonoids

compounds in I. paraguariensis leaves. In summary, our

Table 3 Effect of genotype, canopy structure, branch position and their interactions on the phenolics content of Ilex paraguariensis leaves at the

end of the second flash of growth

Cultivar Branch position CHLA 3,4-CQA 3,5-CQA 4,5-CQA CAA RUT

V-6 Interior 49.5 ± 0.4 19.0 ± 0.1 36.3 ± 0.4 50.3 ± 0.8 0.73 ± 0.03 27.3 ± 0.2

Periphery 51.0 ± 0.4 20.4 ± 0.1 56.9 ± 0.6* 59.6 ± 0.6* 0.49 ± 0.01* 43.4 ± 0.5*

V-16 Interior 52.0 ± 0.3 19.2 ± 0.1 40.7 ± 0.2 47.7 ± 0.5 0.53 ± 0.001 30.0 ± 0.2

Periphery 43.1 ± 0.3* 17.3 ± 0.1 62.5 ± 0.4* 55.0 ± 0.3* 0.44 ± 0.004* 44.7 ± 0.4*

SI-49 Interior 38.6 ± 0.2 28.0 ± 0.3 54.3 ± 0.3 65.5 ± 0.6 0.72 ± 0.005 39.4 ± 0.4

Periphery 47.8 ± 0.3* 17.9 ± 0.1* 47.7 ± 0.5* 44.2 ± 0.3* 0.43 ± 0.004* 62.5 ± 0.4*

Data are expressed as mg g-1 of lyophilized dry weight. The mean values are obtained from four samples (n = 4)

CHLA chlorogenic acid, 3,4-CQA 3,4-dicaffeoylquinic acid, 3,5-CQA 3,5-dicaffeoylquinic acid, 4,5-CQA 4,5-dicaffeoylquinic acid, CAA caffeic

acid, RUT rutin

* Significant difference between branch position at P \ 0.05 (t tests)

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results provide evidences which support the hypothesis that

the physiological status of the stock plant at the time that

cuttings are excised is of utmost importance for the sub-

sequent rooting of I. paraguariensis cuttings. The influence

on total soluble phenolics content of different irradiances

given to the stock plants negatively affects the rooting

process since the product of its oxidation cause the

browning and death of the cuttings, therefore, further

physiological/biochemical studies are still needed in this

specie to understand the detailed mechanism of this mor-

phogenetic process.

Author contributions J. Tarrago and R. Fillip performed

the experiments. L. Mroginski and P. Sansberro designed

and instructed the research work. P. Sansberro wrote the

manuscript.

Acknowledgments The authors are gratefully indebted to the sup-

porting funding from SGCyT-UNNE (PI A014), CONICET (PIP

0734), Establecimiento La Cachuera S.A., and Establecimiento Las

Marıas S.A. We extend our deep appreciation to anonymous

reviewers for their critical comments.

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